If you've been checking my blog you'll know that I'm still fussing around with growth conditions for GFAJ-1. Before doing any more work I want to check with you to make sure we agree that it's worth proceeding. (I'm off at a Gordon Conference this week. Various batches of cells are in the incubator, so I may have more growth results when I get back.)
Basically, I haven't observed cell growth in the liquid version of the AML60 medium described by Wolfe-Simon et al.; instead the cells just very slowly die. But the cells grow very well when this medium is solidified with agar. They don't grow if it's solidified with agarose, so I think the agar is providing a missing nutrient. The cells grow very well in liquid AML60 if I supplement it with Casamino Acids (hydrolysed casein) or with an amino acid mixture we use in our competence induction medium. Neither of these supplements can be used in my growth experiments because they contain too much phosphate, so I'm going to test addition of individual amino acids - initially just glutamate and aspartate since they're what's in our competence mix.
An additional complication is that my version of AML60 medium is still not exactly what Wolfe-Simon et al. described: First, the trace element mix I'm using doesn't contain tungsten (Oremland's lab adds 45 nM sodium tungstate to their minimal media, but there's no evidence that GFAJ-1 needs this); I've ordered some, and I'll add it when I get back. Second, I realized that their version of the basic AML60 salts lacked potassium, so I'm adding 10 mM KCl to my medium.
I'm confident that I can find a suitable growth condition for the ±P/±As growth experiments. But this probably won't be exactly the same condition reported by Wolfe-Simon et al., so we need to decide whether it's appropriate to use my growth condition to test their observation.
Here's what I would do:
- Test growth on medium with and without each of my candidate supplements (single amino acids), with and without added phosphate. (At present all my media have 1.5 mM phosphate added.) I want to see that cells grow to high density with at least one of the supplements, but only when phosphate is provided. (If they grow well without any supplement, that's even better.)
- Choose the supplement that gives the fastest growth or the highest growth yield. Do careful growth curves with and without the supplement, with and without 1.5 mM phosphate. Perhaps test intermediate phosphate levels too (3 µM and 30 µM?), since I don't know how much contaminating phosphate my medium might contain. I would monitor growth by plating dilutions on supplemented AML60 agar, and perhaps by flow cytometry (the flow-cytometer person might not be willing to count cells in 40mM arsenic), as microscopic counting turns out to be a pain in the butt. The goal is to get publishable data (1) showing that the cells won't grow in the specified medium, and (2) establishing suitable phosphate-limited growth conditions for the arsenic test. If the cells don't grow at all in supplemented medium with no added phosphate I'll use medium with 3 µM phosphate added.
- Prepare DNA from the phosphate-replete and phosphate-limited cultures. Use PCR and sequencing to check that the strain I'm using really is GFAJ-1. Send you samples of the DNA and the culture media, so you can confirm that your mass-spec assays will work. You have yet to tell me how much DNA you will need; to get enough DNA I might need to grow a scaled-up version of the phosphate-limited culture.
- Again grow cells in the supplemented AML60 medium, phosphate-replete and phosphate-limited, this time with and without 40 mM sodium arsenate added to the media.
- Grow a scaled-up culture of the cells in 40 mM arsenate plus limited phosphate, and prepare DNA. Send you samples of the media and the DNA.
Do you think that this would be seen as a valid test of the reported result? If not I'll probably stop now, as I have lots of other work to do.